Fluorescence in situ hybridization (FISH) has evolved into a powerful tool for biological research and medicine since the 1980s. FISH is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes. (Langer-Safer, et al. 1982 Proc. Natl. Acad. Sci. U.S.A. 79 (14): 4381-5.) Fluorescent probes and fluorescence microscopy are used in conjunction to identify specific features in DNA or detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells and tissue samples. (Amann, et al. 2008 Nature Reviews Microbiology 6: 339-348.) FISH is an important technique in assisting diagnosis and prognosis of a disease, such as cancer.
Fluorescent probes have become powerful tools for analyzing genes, tissues and cells. (Waggoner 1986 Applications of Fluorescent in the Biomedical Sciences, Eds. Taylor et al., New York: Alan R. Liss, Inc. pp. 3-28; Mason, editor 1993 Fluorescent and Luminescent Probes for Biological Activity, Biological Techniques Series, edited by Sattelle, Academic Press Limited, London.) Among others, commonly labeled biomolecules include nucleotides, oligonucleotides, nucleic acids, amino acids, peptides and polypeptides, proteins, carbohydrates and lipids. (Singer & Ward, 1982 Proc. Natl. Acad. Sci. U.S.A. 79:7331-7335; Singer et al. 1986 BioTechniques 4:230-250; Pitta et al. 1990 Strategies 3: 33; Southern, 1975 J. Mol. Biol. 98: 503-517; Alwine et al. 1977 Proc. Natl. Acad. Sci. U.S.A. 74: 5350-5354; Callow et al. 2000 Grenome Res. 10: 2027-2029.)
Previous fluorophore labeling methods used NTP (nucleoside triphosphate) dyes for labeling. (See, e.g., WO 2000/06773). Another fluorescent labeling technique used platinum compounds for nucleic acid labeling (See, e.g., EP1373572B1). The platinum-based labeling compounds attach to the target biomolecules via coordination of the platinum (II) metal center with nitrogen or sulfur atoms on the target biomolecule. The disadvantages of these strategies include that only a limited number of dyes can be introduced into the probes, thus limiting “brightness” of the probes. In addition, the NTP dyes can result in low yield due to poor incorporation of the highly-modified triphosphates, and the platinum-based compounds can cause degradation of the probes. Moreover, both strategies result in nucleobase modifications throughout the strand, including base pair regions that can negatively affect hybridization.
Thus, there remains an unmet need for novel fluorescent probes and fluorophore labeling methodologies that address the shortcomings of existing fluorescent probes and methods.